The present invention, in some embodiments thereof, relates to chemical conjugates and their use in therapy and diagnosis and, more particularly, but not exclusively, to chemical conjugates of a polymer, an anti-angiogenesis agent and a targeting moiety, which are useful, for example, in the treatment and monitoring of bone related diseases and disorders such as bone cancer and bone metastases.
Osteosarcoma is the most common type of primary bone cancer and classified as a malignant mesenchymal neoplasm in which the tumor directly produces defective osteoid (immature bone). It is a highly vascular and extremely destructive malignancy that most commonly arises in the metaphyseal ends of long bones. Over the past two decades, multimodality treatment consisting of aggressive chemotherapy combined with radical surgical resection, has been the mainstay of osteosarcoma management, with achievable 5 year survival rates of 50% to 70% in patients who do not have metastatic disease at presentation. Several strategies were proposed, such as immune-based therapy, tumor-suppressor or suicide gene therapy, or anticancer drugs that are not commonly used in osteosarcoma [Quan et al. Cancer Metastasis Rev 2006; 10: 707-713]. However, still one-third of patients die from this devastating cancer, and for those with unresectable disease there are no curative systemic therapies.
Prostate cancer is the most common cancer of males in industrialized countries and the second leading cause of male cancer mortality. Mortality in these patients is not due to primary tumor growth, but rather due to complications caused by metastases to vital organs. Prostate cancer predominantly metastasizes to bone, but other organ sites are affected including the lung, liver, and adrenal gland.
Breast cancer also often metastasizes to bones.
Bone metastases incidence in patients with advanced metastatic disease is approximately 70%. Bone metastases are associated with considerable skeletal morbidity, including severe bone pain, pathologic fracture, spinal cord or nerve root compressions, and hypercalcemia of malignancy. Chemotherapy agents, hormonal deprivation and bisphosphonates are the common treatments for advanced metastatic disease. However, with time, the disease progresses to a phase when the standard therapy fails to control the malignancy and further progresses to a highly chemotherapy-resistant state.
Tumor progression and metastases are highly dependent on oxygen and nutrients supplied by new blood vessels, which formation is stimulated by the tumor itself and its environment. Anti-angiogenic therapy combined with conventional treatment holds great potential for osteosarcoma management and metastatic risk reduction. Angiogenesis inhibitors, such as TNP-470 [Folkman, J. Apmis 2004; 112: 496-507], its non-toxic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-conjugated form, caplostatin [Satchi-Fainaro et al. Cancer Cell 2005; 7: 251-261], and Avastin [Hurwitz et al. N Engl J Med 2004; 350: 2335-2342] are emerging as a new modality of anticancer drugs.
There are currently eight approved anti-cancer therapies with recognized antiangiogenic properties. These agents, which interrupt critical cell signaling pathways involved in tumor angiogenesis and growth, can be divided into two primary categories: (1) monoclonal antibodies directed against specific proangiogenic factors and/or their receptors; (Avastin, Erbitux, Vectibix, Herceptin) and (2) small molecule tyrosine kinase inhibitors (TKIs) of multiple proangiogenic growth factor receptors (Tarveca, Nexavar, Sutent). Inhibitors of mTOR (mammalian target of rapamycin) represent a third, smaller category of antiangiogenic therapies with one currently approved agent (Torisel). In addition, at least two other approved anti-angiogenic agents may indirectly inhibit angiogenesis through mechanisms that are not completely understood (Velcade, Celgene)
The first FDA-approved angiogenesis inhibitor, Bevacizumab (Avastin, Genentech), a monoclonal antibody to vascular endothelial growth factor (VEGF), has recently been approved for metastatic colon cancer treatment in conjunction with standard conventional chemotherapy.
The largest class of drugs that block angiogenesis are the multi-targeted tyrosine kinase inhibitors (TKIs) that target the VEGF receptor (VEGFR). These drugs such as sunitinib (Sutent, Pfizer), Sorafenib (Nexavar, Bayer/Onyx Pharmaceuticals) and Erlotinib (Tarveca, Gennentech/OSI/Roche) have the advantages of hitting multiple targets, convenient oral administration, and cost effectiveness. While these drugs exhibit promising efficacy, their use is limited by their lack of target specificity, which leads to unexpected toxicity [Cabebe et al. Curr Treat Options Oncol 2007; 8:15-27].
Novel targeted angiogenesis inhibitors, for use with or without other anti-neoplastic agents have therefore been sought for. A major impediment towards this effort has been the inability to determine therapeutic efficacy, the lack of reliable surrogate markers of tumor angiogenesis, and the complexity of interactions between multiple host cells and malignant cells involved in tumor angiogenesis, which may limit the use of a single anti-angiogenic agent. Another significant obstacle is that the vast majority of clinically used anti-cancer and anti-angiogenic drugs are small molecules that exhibit a short half-life in the bloodstream and a high overall clearance rate. These low-molecular weight drugs diffuse rapidly into healthy tissues and are distributed evenly within the body. As a consequence, relatively small amounts of the drug reach the target site, and therapy is associated with low efficacy and severe side effects.
TNP-470 is a low molecular weight synthetic analogue of fumagillin, which is capable of selectively inhibiting endothelial growth in vitro. In clinical trials, this drug was found to slow tumor growth in many patients with metastatic cancer and exhibited a promising efficacy when used in combination with conventional chemotherapy. However, at the doses required for tumor regression, many patients experienced neurotoxicity. Due to its dose-limiting neurotoxicity, no further clinical studies were conducted for using TNP-470 per se. It has been concluded that clinical uses of TNP-470 should be performed with this agent being targeted to tumor tissue, in order to increase its site specificity and reduce side effects.
Water-soluble polymers such as N-(2-Hydroxypropyl)methacrylamide copolymers (HPMA) are biocompatible, non-immunogenic and non-toxic carriers that enable specific delivery into tumor tissue [Satchi-Fainaro et al. Nat Med 2004; 10: 255-261]. These macromolecules do not diffuse through normal blood vessels but rather accumulate selectively in the tumor site because of the enhanced permeability and retention (EPR) effect. This phenomenon of passive diffusion through the hyperpermeable neovasculature and localization in the tumor interstitium is observed in many solid tumors for macromolecular agents and lipids. Conjugation of anti-cancer drugs such as TNP-470 with copolymers, such as HPMA, should enable selective targeting of these drugs to tumor tissue and thus reduce side effects. Furthermore, such copolymer-drug conjugates should restrict the passage through the blood brain barrier and would prolong the circulating half-life of the drugs, hence inhibiting the growth of tumor endothelial and epithelia cells by exposing the cells to the conjugated drugs in the circulation for a longer time compared to the free drugs.
An example of the favorable characteristics obtained by conjugation of an anti-angiogenesis agent such as TNP-470 to HPMA has been described by Satchi-Fainaro et al. in WO 03/086382. This patent application teaches conjugates of water-soluble polymers and TNP-470, and their use as anti-tumor agents, in particular their use as carriers of TNP-470 into tumor vessels, and their effect on the neurotoxicity of TNP-470. According to the teachings of WO 03/086382, an exemplary such conjugate, HPMA-(TNP-470) conjugate (caplostatin), exhibited superior antitumor activity together with a reduced level of toxicity, as compared with TNP-470 alone. WO 03/086382 further suggests incorporation of a targeting ligand, such as RGD or antibodies.
The use of HPMA-TNP-470 conjugate for the treatment of angiogenesis related conditions has also been described in WO 03/086178.
Another example of the increased activity yet reduced toxicity obtained by conjugation of anti-tumor drugs to water-soluble polymers is presented in U.S. Pat. No. 6,884,817.
An HPMA copolymer conjugate of paclitaxel has been described by Meerum Terwogt et al. [Anticancer drugs 2001; 12:315-323]. This conjugate was aimed at improving drug solubility and providing controlled release of paclitaxel. In this conjugate, the paclitaxel is linked to the HPMA copolymer through an ester bond, and is hence released from the polymer by non-tissue specific hydrolytic or enzymatic (esterases) degradation of the ester bond, thereby inducing the commonly observed toxicities of paclitaxel.
Bisphosphonates (BPs) such as alendronate are compounds with a chemical structure similar to that of inorganic pyrophosphate (PPi), an endogenous regulator of bone mineralization. Several bisphosphonates are established as effective treatments in clinical disorders such as osteoporosis, Paget's disease of bone, myeloma, and bone metastases. Bisphosphonates, such as zoledronic acid, have been shown to inhibit angiogenesis [Wood et al. J Pharmacol Exp Ther 2002; 302: 1055-1061]. The pharmacokinetic profile of bisphosphonates, which exhibit a strong affinity to bone mineral under physiological conditions, their low toxicity and anti-angiogenic activity are advantageous for targeting to tumors confined to bony tissues.
Alendronate is considered potent for the treatment of bone related diseases and cancer-associated hypercalcemia. It was shown to have antitumor effect in several in vivo cancer models through several different mechanisms [Tuomela et al. 2008, BMC Cancer 8:81; Molinuevo et al. 2007, Eur J Pharmacol 562:28-33; Hashimoto et al. 2005, Cancer Res 65: 540-545]. In addition, alendronate was found to have anti-angiogenic activity through (i) suppression of VEGF-induced Rho activation in an ovarian cancer model [Hashimoto et al. 2007, Biochem Biphys Res Commun 354: 478-484], (ii) inhibition of farnesyl pyrophosphate synthase, in the mevalonate pathway [Russell R G 2007, Pediatrics 119 Suppl 2: S150-162]; and (iii) regulation of cellular level of MMP-2 expression in osteosarcoma cell lines [Cheng et al. 2004, Pediatr Blood Cancer 42; 410-415].
Other bone targeting agents are oligopeptides of Aspartate. Wang et al. describe fluorescein-labeled bone-targeted model conjugates for detection purposes bearing 1% loading of D-Asp8 (SEQ ID NO:1) on HPMA copolymer [Wang et al. 2003 Bioconjug Chem 14:853-859]. The bone-targeting potential of this conjugate was tested in vitro and in vivo and was found to selectively accumulate in bone tissue [Wang et al. 2006, Mol Pharm 3:717-725].
WO 2004/062588 teaches water soluble polymeric conjugates for bone targeted drug delivery with improved pharmacokinetics parameters and better water solubility of the loaded drugs. The polymeric drug delivery systems taught by this application are based on hydroxypropyl methacrylamide (HPMA) conjugates of bone-targeting drugs such as alendronate and D-Asp8 together with a bone-related therapeutic agent. The loading of alendronate and D-Asp8 (SEQ ID NO:1) onto the HPMA copolymer was 0.494 mmol/gram and 0.762 mmol/gram respectively.
PK2 (FCE28069) is a HPMA copolymer-doxorubicin-galactosamine conjugate, which was designed as a treatment for hepatocellular carcinoma or secondary liver disease [Seymour et al. Journal of Clinical Oncology 2002; 20:1668-1676]. Doxorubicin is an anthracycline antibiotic with limited solubility in physiological fluids, and is a well established anti-neoplastic drug. Galactosamine binds to the hepatic asialoglycoprotein receptor (ASGPR) thus serving as a specific hepatic targeting moiety. These components are linked to the HPMA polymer via an enzymatically biodegradable linker which permits the release of free doxorubicin within the liver, thus increasing the drug concentration in its site of action. The enzymatic degradable linker is a tetrapeptide spacer (Gly-Phe-Leu-Gly) (SEQ ID NO:2), designed for cleavage by lysosomal cathepsins.
O'hare et al. [Journal of Drug Targeting 1993; 1:217-229] have synthesized HPMA copolymers containing doxorubicin and melanocyte stimulating hormone (MSH) as a melanoma specific targeting moiety. Both the doxorubicin and the melanocyte stimulating hormone were linked to the HPMA polymer via an enzymatically biodegradable linker.
Hruby et al. [Journal of Applied Polymer Science 2006; 101: 3192-3201] have prepared and synthesized novel polymeric drug-delivery systems designed for bone targeting of anti-neoplastics based on biocompatible HPMA copolymers containing hydroxybisphosphonate targeting moieties and the model drugs radiotherapeutics 125I, imaging agent 111In, or the anticancer drug Doxorubicin. The percentage of hydroxybisphosphonate loaded onto the HPMA copolymers was in the range of 1.3-4 mol %.